RNA Extraction Kit
QuickExtract™ FFPE RNA Extraction Kit
- Isolation of RNA from archived FFPE samples for RT-PCR-based analysis.
The QuickExtract™ FFPE RNA Extraction Kit provides a fast, simple, and inexpensive method for preparing RNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples for RT-PCR or real-time RT-PCR (Fig. 1). The kit requires only heat treatment to melt the paraffin, lyse cells, decrease formalin-induced cross-linking, and degrade compounds that may inhibit amplification. Optional DNase reagents are included for use in some downstream applications. Following heat treatment, the RNA sample is ready for RT-PCR.
While RNA from FFPE samples is often degraded and of poor quality, we have demonstrated that multiple transcripts can be extracted (Fig. 2) and that full-length coverage of the desired transcript is also possible (Fig. 3). The protocol is ideal for high-throughput applications, with minimal hands-on time required to process the sample. An optional protocol allows for simultaneous extraction of both RNA and DNA from the sample.
QuickExtract™ RNA Extraction Kit
- RT-PCR-ready extracted RNA in 30 minutes, ideal for high-throughput applications.
- No xylene or phenol extractions.
- No columns, transfers, or sample loss.
- Optional protocol allows simultaneous extraction of RNA and DNA from the same tissue sample.
Figure 1. Overview of the QuickExtract™ FFPE RNA extraction procedure.
|Figure 2. RT-PCR amplification of a series of different messages present in skeletal muscle RNA. RNA was extracted from slide-mounted, FFPE human tissue samples per the protocol and reverse transcribed with the MMLV Reverse Transcriptase 1st Strand cDNA Synthesis Kit and random primers. Skeletal muscle cDNA was amplified to produce short amplicons from a number of different messages. The products were separated on a 3% agarose gel and were visualized with SYBR® Gold. Lane M, 100-bp DNA ladder; lane 1, a 116-bp region of RYR1; lane 2, a 162-bp region of ACTA; lane 3, a 166-bp region of RSP18; lane 4, a 166-bp region of ENO3; lane 5, a 226-bp region of GAPDH; lane 6, a 232-bp region of OAZ1; lane 7, a 307-bp region of ACTB; lane 8, a 308-bp region of TNF.||Figure 3. PCR amplification of five regions of the 15.4-kb ryanodine receptor 1 (RYR1) cDNA. RNA was extracted as per the protocol from FFPE slide-mounted human skeletal muscle tissue samples and reverse-transcribed with the MMLV Reverse Transcriptase 1st Strand cDNA Synthesis Kit and random primers. The RYR1 PCR products were separated on a 3% agarose gel and were visualized with SYBR® Gold. Lane M, 100-bp DNA ladder; RYR1 regions amplified: lane 1, 123-228; lane 2, 2,866-2,987; lane 3, 9,132-9,279; lane 4, 11,842-11,979; lane 5, 14,935-15,097.|
QuickExtract™ RNA Extraction Kit
- Preparation of RNA from cultured adherent and suspension cells for RT-PCR.
The QuickExtract™ RNA Extraction Kit is a fast, simple way of preparing RNA for RT-PCR (both end-point and real-time). The single-tube system requires only vortex mixing to lyse the cells, and prepare the RNA for cDNA synthesis. The result is easy processing of one to hundreds of samples in minutes, with no sample loss or toxic organic solvents. The QuickExtract RNA Extraction Solution works with cultured adherent and suspension cells including buccal cells, and has been tested on human, mouse, rat, E. coli, and S. aureus cell cultures. It is not suitable for tissue samples or plant samples. An optional DNase I treatment may improve certain downstream applications.
- Single-tube system.
- Prepare lysate in minutes.
- No toxic organic solvents.
- Suitable for high-throughput applications.
- Optional DNase I treatment.
||Figure 1. End-point RT-PCR of different regions of a 14 kb message using HeLa cell extract with the QuickExtract™ RNA Extraction Kit. A sample containing 105 HeLa cells was lysed in 100 µl of QuickExtract RNA Extraction Solution by vortex mixing. The lysate was reverse transcribed with the MMLV Reverse Transcriptase 1st Strand cDNA Synthesis Kit using standard conditions and random primers. The cDNA was then amplified with six primer sets to p532 using the FailSafe™ PCR System. Lane M, 100 bp ladder; Lane 1, 12984 to 13892; lane 2, 9406 to 10202; lane 3, 5194 to 5802; lane 4, 4191 to 4690; lane 5, 2280 to 2676; and lane 6, 1029 to 1329.|
||Figure 2. Comparative yield of RT-PCR product with different RNA extraction kits. Lysates were prepared according to manufacturers' instructions and used as template to produce cDNA using the MMLV RT 1st Strand cDNA Synthesis Kit, followed by PCR using the FailSafe™ PCR System with primers for the ALDOA gene. Products were separated on a 2% agarose gel and stained with SYBR® gold. Lane M, 100 bp ladder; lanes 1 and 2, QuickExtract™ RNA Extraction Kit; lanes 3 and 4, kit from Vendor 1; lanes 5 and 6, kit from Vendor 2.|